The Ginkgo biloba tree is among the oldest living plants and is referred to as a “living fossil”. It was introduced into the Western world in the 18th century and admired for its unique beauty, especially the leaves, as exemplified by a 1815 poem by Wilhelm von Goethe referring to its beauty (Goethe, 1819). The Ginkgo tree has a long history of use in traditional Chinese medicine, but it was not until the 1960's that a standardized extract of G. biloba leaves, EGb 761, was introduced into the European markets (Defeudis, 1998). Today G. biloba extract is one of the most popular botanical medicines worldwide.
Numerous beneficial effects of EGb 761 has been postulated including improving neuroprotection in Alzheimer's disease. Alzheimer's disease is a progressive, inexorable loss of cognitive function associated with an excessive number of senile plaques in the cerebral cortex and subcortical gray matter, which also contains β-amyloid and neurofibrillary tangles consisting of tau protein. The cause of Alzheimer's disease is not known (Merck Manual, 1999).
EGb 761 has also been postulated in improving peripheral vascular function, inhibition of thrombosis and embolism cognitive disorders, anti-inflammatory and antiproliferative activities, as well as antioxidant activities (Defeudis, 2000). EGb 761 is a complex mixture of compounds, the main ingredients being flavonoids and terpene trilactones (ginkgolides and bilobalide) that comprise 24% and 6%, respectively, of the total extract (Ahlemeyer, 1998; Drieu, 1986). It is considered that the flavonoids act as antioxidants, while the terpene trilactones are involved in anti-inflammation and prevention of blood clotting associated with the antagonistic activity at the platelet-activating factor (PAF) receptor (McKenna, 2001). However, the neuroprotective effects of EGb 761 have so far not been associated with specific components of the extract (Peskind, 1998; Simonson, 1998).
The terpene trilactones, the ginkgolides (FIG. 1) and bilobalide, are unique components of EGb 761, the structures of which were elucidated in 1967 (Maruyama, 1967; Maruyama, 1967; Maruyama, 1967; Maruyama, 1967; Woods, 1967; Nakanishi, 1967; Okabe, 1967). The ginkgolides are diterpene trilactones with a cage-like skeleton consisting of six 5-membered rings, i.e., a spiro[4.4]nonane carbocyclic ring, three lactones, and a tetrahydrofuran moiety. Terpene trilactones from G. biloba are also among the very few natural products containing a tert-butyl group.
In contrast to many studies on the neuroprotective effects of EGb 761, the ginkgolides have not been extensively studied partly due to limited availability of pure ginkgolides. The finding in 1985 that ginkgolide B (GB, 2) was a potent antagonist of the PAF receptor (PAFR) (Braquet, 1986; Braquet, 1985) led to extensive structure-activity relationship (SAR) studies on this receptor (Rapin, 1998; Braquet, 1987; Braquet, 1991; Hu, 2001; Hu, 2000; Hu, 2000; Corey, 1991; Park, 1993; Park, 1995; Stromgaard, 2002). However, the significance of these effects in relation to the neuroprotective effects of EGb 761 is not clear (Stromgaard, 2003).
The first indication of a direct interaction of ginkgolides with important targets in the brain was discovered when ginkgolides were shown to be potent and highly selective antagonists of the inhibitory glycine receptor (GlyR) (Stromgaard, 2003; Kondratskaya, 2002). The GlyR is a ligand-gated ion channel found primarily in spinal cord and brain stem, but also in higher brain regions such as hippocampus and developing cortex that consists of α1-α4 and β subunits (Betz, 2001). Only a few ligands for GlyRs have been available, the classical example being the convulsant strychnine, a competitive antagonist. However, since the neuropharmacology and functional importance of GlyRs in higher brain regions is not well characterized (Chattipakorn, 2002) new potent and selective ligands are needed for further investigations of the GlyR.
The introduction of combinatorial chemistry and application of related techniques such as parallel or split-and-pool synthesis, solid and solution-phase strategies have opened avenues to libraries of various compounds (Marcaurelle, 2002; Arya, 2001; Ganesan, 2002; Abel, 2002; Krchnak, 2002; Schreiber, 2000). These compounds could be entirely artificial or derivatives of natural products with possible enhanced bioactivities. Recently diversity-oriented semi-synthetic methods have been used for the preparation of library of natural product derivatives for biological activity studies (Abel, 2002). In the following we describe a parallel solution-phase synthesis of a ginkgolide combinatorial library. Diversity is achieved by modification of traditional synthetic methods to prepare novel ginkgolide derivatives in a selective fashion. The ginkgolide derivatives have been evaluated as antagonists of the GlyR using a fluorescence-based high-throughput screening assay.